Two potential clinical applications of the alkaline single-cell gel electrophoresis assay .1. Human bladder washings and transitional cell carcinoma of the bladder .2. Human sperm and male infertility

Part 1: The alkaline single-cell gel electrophoresis (comet) assay was used to analyse the integrity and DNA content of exfoliated cells extracted from bladder washing specimens from 9 transitional cell carcinoma patients and 15 control patients. DNA damage, as expressed by % tail DNA and tail moment values, was observed to occur in cells from both control and bladder cancer samples. The extent of the damage was, however, found to be significantly greater in the cancer group than in the control group. Comet optical density values were also recorded for each cell analysed in the comet assay and although differences observed between tumour grades were not found to be statistically significant, the mean comet optical density value was observed to be greater in the cancer group than in the control population studied, These preliminary results suggest that the comet assay may have potential as a diagnostic tool and as a prognostic indicator in transitional cell carcinoma, Part 2: Baseline DNA damage in sperm cells from 13 normozoospermic fertile males, 17 normozoospermic infertile males and 11 asthenozoospermic infertile males were compared using a modified alkaline comet assay technique. No significant difference in the level of baseline DNA damage was observed between the 3 categories of sperm studied; however the untreated sperm cells were observed to display approximately 20% tail DNA. This is notably higher than the background DNA damage observed in somatic cells where the % tail DNA is normally less than 5%. Sperm from the 3 groups of men studied were also compared for sensitivity to DNA breakage, using the modified alkaline comet assay, following X-ray irradiations (5, 10 and 30 Gy) and hydrogen peroxide treatments (40, 100 and 200 mu M). Significant levels of X-ray-induced damage were found relative to untreated control sperm in the two infertile groups following 30 Gy irradiation. Significant damage in hydrogen peroxide-treated sperm was observed in sperm from fertile samples, at 200 mu M and in infertile samples at 100- and 200-mu M doses relative to controls. These results therefore indicate that fertile sperm samples are more resistant to X-ray- and hydrogen peroxide-induced DNA breakage than infertile samples. Further studies involving greater numbers of individuals are currently in progress to confirm these findings.

DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.

Inherited resistance to arsenate toxicity in two populations of Lumbricus rubellus

No unequivocal evidence exists of genetically inherited resistance to metals/metalloids in field populations of earthworms. We studied cocoon production in adult Lumbricus rubellus Hoffmeister collected from an abandoned arsenic and copper mine (Devon Great Consols, Devon, UK), and abandoned tungsten mine (Carrock Fell, Cumbria, UK) and an uncontaminated cultured population. The earthworms were kept in uncontaminated soil for nine weeks. From a total of 42 L. rubellus from each site, Devon Great Consols adults produced 301 cocoons, of which 42 were viable; Carrock Fell 60 cocoons, of which 11 were viable; and the reference population 101 cocoons, of which 62 were viable. The hatchlings were collected and stored at 4degreesC at weekly intervals. After 12 weeks, all hatchlings were transferred to clean soil and maintained at 15degreesC for 20 weeks until they showed evidence of a clitellum. In toxicity trials, F1 generation L. rubellus were exposed to 2,000 mg As/kg as sodium arsenate or 300 mg Cu/kg as copper chloride for 28 d. The F1 generation L. rubellus from Devon Great Consols mine demonstrated resistance to arsenate but not copper. All L. rubellus from Devon Great Consols kept in soil treated with sodium arsenate remained in good condition over the 28-d period but lost condition rapidly and suffered high mortality in soil treated with copper chloride. The control population suffered high mortality in soil treated with sodium arsenate and copper chloride. Previous work has shown that field-collected adults demonstrate resistance to both arsenate and Cu toxicity under these conditions. Thus, while arsenate resistance may be demonstrated in F1 generation L. rubellus from one of the contaminated sites, Cu resistance is not. The F1 adults and F2 cocoons did not have significantly higher levels of As than the control population, with no residual As tissue burden, suggesting that resistance to As in these populations may be inherited.

Priming and re-drying improve the survival of mature seeds of Digitalis purpurea during storage

Most priming studies have been conducted on commercial seed lots of unspecified uniformity and maturity, and subsequent seed longevity has been reported to both increase and decrease. Here a seed lot of Digitalis purpurea L. with relatively uniform maturity and known history was used to analyse the effects of priming on seed longevity in air-dry storage. Seeds collected close to natural dispersal and dried at 15 % relative humidity (RH), 15 degrees C, were placed into experimental storage (60 % RH, 45 degrees C) for 14 or 28 d, primed for 48 h at 0, -1, -2, -5, -10 or -15 MPa, re-equilibrated (47 % RH, 20 degrees C) and then returned to storage. Further seed samples were primed for 2 or 48 h at -1 MPa and either dried at 15 % RH, 15 degrees C or immediately re-equilibrated for experimental storage. Finally, some seeds were given up to three cycles of experimental storage and priming (48 h at -1 MPa). Priming at -1 MPa had a variable effect on subsequent survival during experimental storage. The shortest lived seeds in the control population showed slightly increased life spans; the longer lived seeds showed reduced life spans. In contrast, seeds first stored for 14 or 28 d before priming had substantially increased life spans. The increase tended to be greatest in the shortest lived fraction of the seed population. Both the period of rehydration and the subsequent drying conditions had significant effects on longevity. Interrupting air-dry storage with additional cycles of priming also increased longevity. The extent of prior deterioration and the post-priming desiccation environment affect the benefits of priming to the subsequent survival of mature seeds. Rehydration-dehydration treatments may have potential as an adjunct or alternative to the regeneration of seed accessions maintained in gene banks for plant biodiversity conservation or plant breeding.

Seleção para peso pós-desmama em um rebanho Gir. 1. Resposta direta e correlacionada em 21 anos de seleção

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Qualidade subjetiva do sono e queixa de insônia em pacientes com Acidente Vascular Cerebral

Brain injury can be associated with changes in the sleep-wake cycle. However, studies about sleep disturbances and their relationship with quality of sleep are scarce. Besides, it remains to be known how stroke affects the mechanisms of sleep. The aim of this study was to investigate quality of sleep, complaints of sleep disturbances and associated factors in stroke patients from the Physical Therapy services in Natal -RN. This was a cross-sectional descriptive study involving 70 individuals (aged 45-65 years), 40 patients (57 ± 7 years), 11 ± 9 months after injury, and 30 healthy individua ls (52 ± 6 years), evaluated with the Pittsburgh Sleep Quality Index (PSQI) and Sleep Habits Questionnaire. The data were analyzed by Chi-square test, t Student test and logistic regression. Poor quality sleep was found in 57,5% of the patients (6,3 ± 3,5) and was significantly higher than in the control population (3,9 ± 2,2) (t Student test, p=0,002). The patients showed significantly higher value of PSQI than controls: sleep latency (p=0,019), length of sleep (p=0,039) and dysfunction during the day (p=0,001). Regarding complaints of sleep disturbances (dyssomnias and parasomnias) analyzed by Chi-square test, the complaint of insomnia was the most prevalent (patients: 37,5%; healthy subjects: 6,7%; p=0,007). Regression analysis showed that sl eep latency (p=0,036) and complaint of insomnia (p=0,036) were associated with quality sleep. In addition, female gender (p=0,036) and complaint of broken sleep (p=0,003) were considered risk factors for the presence of insomnia. Our results show that stroke affects the homeostatic process of sleep. Shorter sleep latency and the absence of insomnia are considered protective factors for good sleep quality and this should be taken into consideration in the diagnostic and therapeutic strategies

Laryngeal and voice disorders in patients with gastroesophageal symptoms. Correlation with pH-monitoring

OBJETIVO: Investigar as alterações laríngeas e vocais em pacientes com sintomas de refluxo gastroesofágico e correlacioná-las com o exame de phmetria. MÉTODOS: Estudo prospectivo que incluiu os pacientes atendidos nos ambulatórios de Distúrbios da Voz da Faculdade de Medicina de Botucatu no período de cinco anos com sintomas vocais e gastroesofágicos. Os pacientes foram submetidos à videolaringoscopia, às análises vocais perceptivo-auditivas, a analise vocal acústica computadorizada e ao exame de pHmetria de dois canais com monitorização durante 24 horas. RESULTADOS: Foram incluídos 57 pacientes (entre 21 a 65 anos; 45 mulheres e 12 homens). Desses, 18 apresentavam pHmetria normal (31,6%) e 39 alterada (68,4%). As videolaringoscopias registraram diversas lesões laríngeas tanto nos pacientes com pHmetria normal como alterada, sendo mais relevantes neste último grupo, destacando-se a paquidermia posterior. As avaliações vocais perceptivo-auditivas identificaram alterações vocais de diversas intensidades em ambos os grupos, mais importantes nos pacientes com pHmetria alterada. Todos os parâmetros acústicos, exceto Fo, mostraram-se alterados em ambos os grupos, quando comparados aos controles. CONCLUSÕES: Alterações vocais perceptivas e acústicas, e lesões laríngeas foram registradas tanto nos pacientes com phmetria normal como alterada, sinalizando para a importância da historia clínica e dos achados videolaringoscópicos no diagnóstico das laringites ácidas.

Ácaros predadores associados ao ácaro-da-erinose da lichia

O objetivo deste trabalho foi identificar ácaros predadores em plantas de lichia e correlacionar o desenvolvimento populacional dessas espécies com o do ácaro-da-erinose da lichia, Aceria litchii. A pesquisa foi desenvolvida no Município de Casa Branca, SP, com árvores adultas, de 12 anos de idade, da variedade Bengal. Mensalmente, de agosto de 2008 a setembro de 2009, foram coletadas folhas para avaliação dos níveis populacionais de A. litchii e de ácaros predadores. Foram registrados 6.557 indivíduos da família Phytoseiidae. A espécie mais abundante foi Amblyseius compositus (42,6%), seguida por Phytoseius intermedius (31,2%), Euseius concordis (14,1%), Amblyseius herbicolus (8,8%) e Iphiseiodes zuluagai (3,3%). O desenvolvimento populacional de A. compositus, E. concordis e I. zuluagai correlacionou-se positivamente com o de Aceria litchii, o que indica relação de predação.

Analysis of the TAX1BP1 gene in head and neck cancer patients

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of hand weeding strip and nitrogen fertilizer on corn plants

The objective of the present research was to evaluate effects of different strip weed control associated with nitrogen fertilizer on corn applied after planting. The experiment was set and conducted in Botucatu, São Paulo State, Brazil, and the hybrid planted was Dekalb 333-B. A completely randomized block design with four replications was used. Experimental plots were disposed as a factorial scheme 2 x 2 x 4, constituted by two types of weeding on row (with or without manual hoeing), two types of weeding on inter-row (with or without manual hoeing), and four nitrogen levels applied after planting (00, 60, 90, and 120 kg ha-1). Plots were composed by six rows with 5 m length. Nitrogen fertilizer was applied at 35 days after emergence (d.a.e). For weed community it was evaluated: weed density, dominancy, frequency, and relative importance. The main weed species were: Brachiaria plantiginea, Amaranthus retroflexus, Bidens pilosa, Cyperus rotunds, Brachiaria decumbens, Euphorbia heterofila, Oxalis latifolia, Acanthospermum hispidum, Commelina benghalensis. It was evaluated corn height at 40 and 100 d.a.e., first ear insertion height at 100 d.a.e., and final grain yield at harvesting. Plants and first ear insertion height were affected when nitrogen fertilizer was not applied. Treatments without weed control showed that weed interfered negatively with plants height. There were no correlation between weeds and nitrogen fertilizer for all parameters evaluated. Parcels without weed showed the highest ear weights and final grain production. Treatments that received nitrogen fertilizer, independently of studied arrangement, provided higher yields.

Ajuste de modelos não lineares e estimativas de parâmetros genéticos para produção de ovos de uma linhagem de poedeiras White Leghorn

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Polimorfismos gênicos e danos no DNA em indivíduos com obesidade mórbida

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Leishmaniose visceral canina: busca ativa de casos e estudo sobre o conhecimento da doença em proprietários de cães na área urbana de Botucatu

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dinâmica populacional de moscas-das-frutas (diptera: tephritidae) antes e após a liberação de diachasmimorpha longicaudata (hymenoptera: braconidae) em área de intersecção de pomar cítrico e mata secundária

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5'-TGC GGC TGA GGA GGA CT-3') and BbasCG1024R (5'-TGC GGC TGA GTG TAG AAC-3') were designed for detecting and monitoring this isolate in the field.